Fig 2: GSK3β enhances the stability of CBX2 protein by phosphorylating USP27X.A Amino acid sequence conservation in different species of the motif in USP27X targeted by GSK3β. B MDA-MB-231 or MCF7 cells were treated with lithium chloride for 6 h, and the cell lysates were analyzed by IB using an antibody against pSer. C HA–USP27X WT or HA–USP27X-S135A was co-transfected with or without Flag-GSK3β into HEK-293T cells. Cell lysates were immunoprecipitated using an anti-HA antibody and then analyzed by IB using a specific antibody against pSer. D Triple immunofluorescence (IF) staining of USP27X(green), GSK3β(red), and nuclei (DAPI, blue) was performed in MCF7 and MDA-MB-231 cells. Scale bar, 10 μm. E Cell lysates from MCF7 and MDA-MB-231 were analyzed by IP using antibodies against USP27X and GSK3β, then subjected to IB analysis. IgG was used as the isotype control. F BT549 cells were transfected with control vector or GSK3β for 36 h and then treated with 20 µM MG132 for 6 h. Cell lysates were immunoprecipitated using an antibody against USP27X and then analyzed by IB. G, H MDA-MB-231 (G) and MCF7 cells (H) were transfected with control siRNA or GSK3β siRNA for 36 h and then treated with 20 µM MG132 for 6 h. Cell lysates were immunoprecipitated using an antibody against USP27X and then analyzed by IB. I HEK-293T cells were transfected with Myc–CBX2, Flag–GSK3β and HA–USP27X-WT or HA–USP27X-S135A. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by IB using an anti-HA antibody. J HEK-293T cells were transfected with His–Ub, Myc-CBX2, Flag–GSK3β, and HA–USP27X WT or HA–USP27X S135A. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by IB using anti-Myc and anti-His antibodies. K BT549 cells were transfected with His–Ub, Myc-CBX2, HA–USP27X WT, or GSK3β siRNA. Cell lysates were immunoprecipitated using an anti-Myc antibody and then analyzed by IB using anti-Myc and anti-His antibodies. L BT549 cells transfected with USP27X WT, S135A, or S135Q mutants were treated with 100 μg/ml CHX, harvested at the indicated times, and treated with IB with anti-CBX2 and USP27X antibodies. Quantification of CBX2 levels relative to Vinculin is shown. M Relative CBX2 level of USP27X WT, S135A or S135Q mutants in BT549. For all panels, data are representative results of three independent experiments. Results were shown as mean ± SD; One-way ANOVA test, ***P < 0.001.

Fig 3: The expression of USP27X in BC tissues is positively correlated with the levels of CBX2.A IB detection of USP27X and CBX2 in BC specimens. B Correlation analysis of USP27X and CBX2 in BC tissues. Statistical analyses were performed with the χ2 test. The Pearson r indicates correlation coefficient. C Representative images of immunohistochemical staining of USP27X and CBX2 on tissue microarray of BC specimens (n = 132). Scale bar, 100 μm, 500 μm. D Relative CBX2 score = CBX2 protein expression/ Internal reference protein expression (Vinculin). Relative USP27X score = USP27X protein expression/ Internal reference protein expression (Vinculin). Data of IHC were analyzed using Pearson correlation by GraphPad Prism (8.3). E, F Kaplan–Meier curves showing overall survival of BC patients divided based on USP27X (E) and CBX2 expression (F) by GraphPad Prism (8.3). For all panels, data are representative results of three independent experiments.

Fig 4: USP27X interacts with CBX2.A–C Cell lysates from MCF7 (A), MDA-MB-231 (B), and BT-549 (C) were analyzed by IP using antibodies against USP27X and CBX2, then subjected to IB analysis. IgG was used as the isotype control. D Triple immunofluorescence (IF) staining of USP27X(green), CBX2(red), and nuclei (DAPI, blue) was performed in MCF7, MDA-MB-231, and BT-549 cells. Scale bar, 10 μm. E Purified Myc-CBX2 was incubated with GST, GST-USP27X or GST-USP27X C87A coupled to glutathione-Sepharose beads. Proteins retained on Sepharose were then subjected to IB with indicated antibodies. Recombinant GST-USP27X and GST-USP27X C87A were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. F Representative images show merged PLA and nuclear (DAPI) channels from PLA experiments. In situ PLA between endogenous USP27X and CBX2. Each red dot represents detection of the USP27X-CBX2 interaction complex. Scale bar, 10 μm. G, H Schematic representation of HA-tagged full-length (FL) USP27X (G), Myc-tagged FL CBX2 (H), and their various deletion mutants. I, J HEK-293T cells were cotransfected with HA-USP27X and Myc-tagged FL CBX2 or its deletion mutants and anti-Myc or HA-coupled magnetic beads for IP analysis of cell lysates followed by IB analysis with HA and Myc antibodies. K, L HEK-293T cells were cotransfected with Myc-CBX2 and HA-tagged FL USP27X or its deletion mutants and anti-Myc or HA-coupled magnetic beads for IP analysis of cell lysates followed by IB analysis with HA and Myc antibodies. For all panels, data are representative results of three independent experiments.

Fig 5: USP27X promotes the proliferation, invasion and metastasis of BC cells by up-regulating CBX2.A, B In MCF7 cells, three independent shRNAs were used to knock down CBX2. CBX2 was overexpressed in BT549. A CBX2 protein expression level was detected by IB analysis, and (B) CBX2 mRNA level was detected by qRT-PCR analysis. C USP27X was knocked down in CBX2-overexpressing MDA-MB-231 cells. CBX2 was knocked down in BT549 cells overexpressing USP27X. Cell growth was examined by colony formation. D Cell treatment with ctrl, two USP27X shRNA (1&2) and USP27X shRNA (1&2) + overexpressed CBX2, leads to a significant decrease and increase of the colony number. E Cell treatment with vector, overexpressed USP27X and overexpressed USP27X + CBX2 shRNA leads to a significant increase and decrease. F The indicated cells were subcutaneously injected (1 × 106 cells per mouse) into nude mice (n = 5) of MDA-MB-231. Tumor growth curve (G) and weight (H) were analyzed. I The indicated cells were subcutaneously injected (1 × 106 cells per mouse) into nude mice (n = 5) of BT549. Tumor growth curve (J) and weight (K) were analyzed. L–N The indicated cells were injected into the mammary fat pad of female NOD-SCID mice (5-6 weeks old, n = 6) of BT549 and MDA-MB-231 (L). The number of pulmonary metastatic nodules was counted and quantified of BT549 (M) and MDA-MB-231 (N). Data are represented as mean ± SD of three independent experiments. ***P < 0.001, 1-way ANOVA with Dunnett’s post test (B, D, E, G, H, J, K, M, N).